The corneal epithelium

The front of your eye is a clear dome called the cornea. The cornea refracts light as it enters the eye to help form a clear image on the retina. It also protects the underlying structures like the iris and lens from the outside world.

On the surface of your cornea sit approximately five layers of epithelial cells. This corneal epithelium is also densely packed with nerves, and is home to a small number of immune cells. 

In order for the cornea to become infected (a disease called keratitis), microbes generally need to cross or bypass the epithelium and enter the lower layer of the cornea, called the stroma. If bacteria make it into the stroma, neutrophils migrate into the cornea in response. Neutrophils can kill bacteria, but they also damage the corneal tissue in the process. This causes opacities that can lead to vision impairment or blindness from scarring. 

At left: mouse cornea diagram. Human corneas have a thicker stroma.

Usually we think of immune cells as the responsible party for managing encounters with pathogenic bacteria, but a healthy corneal epithelium hosts very few immune cells. Most of the corneal immune cells reside in the stroma, with a small population at the base of the epithelial layer. Fortunately, the corneal epithelium is very good at resisting bacteria: in fact, bacteria added to the ocular surface of research animal models are simply cleared away within a day. Related, live bacteria are only occasionally detected on healthy human eyes. 

Because the corneal surface is so highly antimicrobial, many researchers study keratitis by scratching through the epithelium and adding bacteria directly into the stroma. 

In her postdoctoral fellowship at the UC Berkeley School of Optometry, Dr. Kroken worked with novel models developed by Dr. Suzanne Fleiszig's research group where bacteria are forced to interact with corneal epithelial cells first, before they can cause infection. Although these experimental conditions don't always lead to keratitis, they reveal what bacteria are capable of in terms of tissue traversal and colonization--all of which can be ultimately resolved by the corneal epithelium, without any apparent recruitment of neutrophils or other additional immune cells. Therefore, one of the main research aims of the Kroken lab is understanding how epithelial cells win against pathogenic bacteria like Pseudomonas aeruginosa, and whether they accomplish this with or without the help of immune cells. 

Pseudomonas aeruginosa

Pseudomonas aeruginosa is found in water and soil. It is capable of infecting damaged epithelial sites of the body: examples include burned skin, the lungs of patients on ventillators, and contact lens wearers. P. aeruginosa is also of critical concern to cystic fibrosis patients, and is capable of acquiring multiple antibiotic resistances. 

Though initially considered an extracellular pathogen, several research groups have demonstrated that P. aeruginosa is capable of replicating inside epithelial cells, including those of the cornea. How P. aeruginosa can accomplish this is currently another area of investigation by the Kroken Lab. This research aim complements our effort to understand the defenses of corneal epithelial cells: we want to understand specifically what P. aeruginosa is up against, and in turn, what it must do to survive in the cornea.


At right: P. aerguinosa filling the cytoplasm of a HeLa cell. HeLa cells are missing some defenses against intracellular pathogens. 

Pathogenesis of P. aeruginosa toward epithelial cells

P. aeruginosa has a number of virulence factors important for corneal infection. One of these is a type three secretion system (T3SS), which is a molecular syringe that injects toxins directly into target host cells. Paradoxically, the T3SS toxins are involved in keeping P. aeruginosa outside of immune cells like macrophages and neutrophils, but one toxin, ExoS, also enables the bacteria to thrive and replicate inside of epithelial cells. 

Epithelial cells harboring P. aeruginosa grow membrane blebs within which bacteria reside and replicate. These are best studied by live and time lapse imaging, because they are too fragile for conventional fixation methods.

We prefer to image live infection experiments for yet another reason: not every individual bacteria does the same thing while infecting a host. In fact, populations of bacteria diversify in virulence factor expression, which might be important for successfully avoiding host defenses. Imaging allows us to assign a space and time to the activities of individual bacteria, instead of relying only on techniques that average out over a population of bacteria. Those techniques may miss infrequent but important events. 

We try to squeeze every last bit of data out of our images, which includes designing thoughtful computational analyses and batch processing. This helps us study events that occur at a low frequency, and capture enough information to ask and answer questions about them. 

Dr. Kroken's Github repository contains in-progress and published scripts.

Staphylococcus aureus

S. aureus is also a major ocular pathogen with apparent ability to cause keratitis when the corneal epithelium is intact, such as during contact lens wear. We are interested in investigating how this is occurs: specifically, which virulence factors play a major role in killing corneal surface cells and eroding epithelial barrier function. Considering that many of the virulence factors of S. aureus are regulated by quorum sensing, our strategies for understanding how this bacterium damages the corneal epithelium rely on methods other than imaging, such as investigating barrier integrity of corneal epithelial cells grown as multilayers in trans-wells.  

Intravital and ex vivo imaging

As an externally exposed and clear tissue, the cornea is amenable to direct in vivo imaging without the need for surgical placement of imaging windows required of other intravital imaging methods. Additionally, whole eyes are able to be kept in organ culture conditions, and as healthy corneas are avascular, the corneal epithelium can also be studied and imaged ex vivo for hours or days. Many of its antimicrobial defenses remain intact in culture. 

We study live corneas to verify key findings made in vitro and in cell culture models, and to learn more about host-microbe dynamics by watching them unfold.

mT/mG mouse, tdTomato membranes